This invention claims priority of the German patent application 100 43 506.8-52 which is incorporated by reference herein.
The invention concerns a method and an apparatus for laser microdissection of specimen regions of interest of a specimen that is mounted on a specimen holder.
xe2x80x9cMicrodissectionxe2x80x9d refers, in the field of biology and medicine, to a method with which a small piece is cut out from a generally flat specimen (for example cells or a tissue section) with a fine, focused laser beam. The cut-out piece is thus available for further biological or medical (e.g. histological) examinations.
U.S. Pat. No. 5,998,129 describes a method of this kind and an apparatus for laser microdissection. The specimen is arranged on a solid, planar support, for example a polymer support film, that is stretched over a glass specimen slide commonly used in laboratories. The method described operates in two steps. In a first step, a specimen region of interestxe2x80x94on which, for example, a selected cell grouping or a histological section is locatedxe2x80x94is cut out with a laser beam. For that purpose, the cut line of the laser beam describes a complete curve around the specimen region of interest. After cutting, the cut-out specimen region of interest is then still adhering to its substrate or resting on the specimen slide. In a second step, an additional laser shot is therefore directed onto the specimen region of interest, and the specimen region of interest is thereby catapulted in the direction of the laser beam into a collection vessel. Because the cut-out specimen region of interest is catapulted out, the method is therefore referred to in abbreviated fashion among specialists as xe2x80x9claser catapulting.xe2x80x9d
One disadvantage of the method occurs already in the first method step. Shortly before the cut line is completed, the cut-out sample field is joined to the surrounding specimen only by a narrow web. As a result of electrical charging or mechanical stress in the web, at this stage of the cut the cut-out specimen region of interest often swings away, i.e. out of the focal plane of the laser beam or behind the remaining support film. It thereby becomes difficult or even impossible to complete the cutting of the swung-away, cutout specimen region and to transport it away by a laser shot.
A further disadvantage of the method is the fact that in practice, the laser must be defocused for the second method step (laser-induced transport). This means that in laboratory work, for the removal of each individual cut-out sample field the laser each time must be defocused (e.g. by adjusting the height of the specimen stage supporting the specimen), the individual xe2x80x9ctransport shotxe2x80x9d must be executed, and then the laser must be focused again for further processing of the specimen. For the user, especially in the context of laboratory examinations with a large number of cuts, this procedure is cumbersome and time-consuming.
It is therefore the object of the invention to describe a method for laser microdissection which allows a specimen field to be cut out from a specimen in reliable and convenient fashion.
This object is achieved by a method for laser microdissection of specimen regions of interest of a specimen that is mounted on a specimen holder which comprises the following method steps:
a) cutting, with a focused laser beam having a defined cut width, along an incomplete cut line largely enclosing the specimen region of interest, such that there remains between the beginning and end of the cut line a stable web of defined width by way of which the specimen region of interest is joined to the surrounding specimen; and
b) severing the web with a single laser pulse, directed onto the web, of a focused laser beam having a cut width widened as compared to the previous cutting, such that after severing, the specimen region of interest falls down by the action of gravity.
In the context of this method, it proves advantageous if the defined cut width during cutting is much narrower than the cut width of the laser beam when severing the web. The defined cut width during cutting can be generated by attenuating the laser intensity as compared to the laser intensity when severing the web. In this case a laser pulse at the full power of the laser can be generated to sever the web.
The aperture of the laser beam can also be reduced by means of a stop. The cut width of the laser beam on the specimen is thereby modified. It proves particularly effective if the cut width of the laser beam when severing the web corresponds at least to the width of the web. Advantageously, the single laser pulse is directed onto the center of the web.
It is a further object of the invention to provide an apparatus for laser microdissection which allows a sample field to be reliably cut out of a specimen, and which dispenses with any defocusing of the laser beam to carry off the specimen.
This object is achieved by an apparatus for laser cutting of microscopic specimens which comprises a microscope having at least one objective that defines an optical axis and serves for viewing of a specimen having a specimen region of interest, which furthermore comprises a laser that generates a laser beam, and at least one optical system that couples the laser beam into the objective, and which according to the present invention has the following features:
a) a cut line control unit that is associated with the microscope in order to generate a relative movement between the laser beam and the specimen; and
b) means for severing the web, with which the cut width of the laser beam is enlarged and a single focused laser pulse is directed onto the web.
Among specialists it was heretofore generally considered to be impossible to remove a cut-out specimen region of interest from a specimen without laser catapulting. The method according to the present invention now for the first time offers the possibility of cutting out a specimen region of interest and detaching it from the specimen without material-damaging laser bombardment.
For that purpose, laser cutting of the specimen is performed in two method steps with different laser parameters and different laser beam cut widths. Suitable preparation of the specimens is critical for this. For this purpose, the specimens to be examined, from which specimen regions of interest are to be cut out, are prepared on very thin plastic films. The thickness of these plastic films is on the order of between 1 and 2 xcexcm. PET films, for example, can be used. The best cutting results have been obtained, however, with PEN films. With these it is possible to generate a narrow and at the same time stable web. It has proven particularly favorable for the method if the width of the web corresponds to approximately three to five times the cut width of the laser during cutting. The plastic films are stretched, in known fashion, over a specimen holder. This can be, for example, a glass specimen slide commonly used in laboratories. Other specimen holders (in terms of shape and material) are, however, conceivable. The specimen holder rests on an X-Y stage which allows different specimen regions to be viewed and selected. The apparatus usually has at least one vessel, below or in the vicinity of the specimen, for collecting a cut-out specimen region of interest.
One embodiment of the apparatus according to the present invention has a stationary laser beam. The cut line control unit comprises a displaceable X-Y stage which moves the specimen relative to the stationary laser beam during cutting. In this context, very high demands are made on the positioning accuracy of the X-Y stage in order to produce an exact cut line and a web of suitable width. The X-Y stage is preferably displaced in motorized fashion.
In another embodiment of the apparatus according to the present invention, the cut line control unit comprises a laser scanning device which moves the laser beam relative to a stationary specimen during cutting. For that purpose, the X-Y stage with the specimen holder and specimen on it is not displaced during cutting. The cut line results exclusively from deflection of the laser beam over the specimen.
A particularly advantageous embodiment of the apparatus is one in which a laser control unit which controls the operating parameters of the laser is associated with the laser. Those operating parameters are, for example, the laser power and the laser aperture, which determine the laser cut width. An autofocus apparatus for the laser can additionally be provided; for a clean cut, it ensures reliable focusing even with specimens of different thicknesses.
In another advantageous embodiment, a computer which is used to control the cut line control unit and the laser control unit is associated with the microscope. Automation of the entire method is thereby possible.
In other embodiments of the apparatus, means for selection of the cut line, or means for selection of the cut line and the position of the web by a user, are provided. In addition, means can be provided for selection of the width of the web and for selection of the position of the web by a user. By way of this selection capability, the user can specifically select the correct specimen region of interest before cutting, and at the same time can protect important portions of the specimen from damage. Because the user can, for example, place the cut line on non-critical cell structures of the specimen, cell structures of interest within the specimen region of interest can be protected during cutting.
In a further embodiment, means for automatic enlargement of the cut width of the laser beam and for automatic execution of a single laser pulse, directed onto the web, with that cut width, are associated with the microscope.
The method according to the present invention possesses the advantage of ruling out any swinging away of the specimen region of interest during cutting. Problem-free cutting of the specimen is thereby possible. In addition, severing of the web simultaneously makes possible reliable detachment from the specimen of the cut-out specimen region of interest. The cut-out specimen region of interest then needs only to be collected. Automation of the method and of the apparatus makes possible utilization in routine laboratory operations.
The essential advantage of the method consists, however, in the fact that both cutting and severing of the web are accomplished with a laser beam focused on the cut line and the web, respectively. As a result, the specimen region of interest enclosed by the cut line is protected, during both method steps, from possible damage due to laser irradiation. In this fashion, in contrast to previously known methods, no laser radiation is directed onto the specimen region of interest. This is a very important aspect specifically in the context of biological specimens, since radiation-related changes in cell structures or in genetic information inside the specimen region of interest can in this fashion be ruled out. In addition, it proves advantageous for laboratory operation that the method according to the present invention operates without repeated and therefore time-consuming focusing and defocusing.